Comparison of SARS-CoV-2 Detection from Saliva Sampling and Oropharyngeal Swab

ABSTRACT We examined the detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse transcription-PCR (RT-PCR) of side-by-side saliva and oropharyngeal swab (OPS) samples from 639 symptomatic and asymptomatic subjects, of which 47 subjects were found to be positive for SARS-CoV-2 in the OPS or saliva sample or both. It was found that the detection rate (93.6% for both OPS and saliva) as well as the sensitivity and specificity were comparable between the two sampling methods in this cohort. The sensitivity was 0.932 (95% confidence interval [CI], 0.818 to 0.977) and the specificity was 0.995 (95% CI, 0.985 to 0.998), both for saliva when OPS sampling was used as the reference and for OPS when saliva was used as the reference. Furthermore, the Cohen’s kappa value was 0.926 (95% CI, 0.868 to 0.985), indicating strong agreement between the two sampling methods. In addition, the viral RNA stability in pure saliva and saliva mixed with preservation buffers was examined following storage at room temperature and at 4°C. It was found that pure saliva kept the viral RNA stable for 9 days at both temperatures and that the type of preservation buffer can either enhance or reduce the stability of the RNA. We conclude that self-administered saliva sampling is an attractive alternative to oropharyngeal swabbing for SARS-CoV-2 detection, and it might be useful in large-scale testing. IMPORTANCE It is not inconceivable that we will witness recurring surges of COVID-19 before the pandemic finally recedes. It is therefore still relevant to look for feasible, simple, and flexible screening methods so that schools, workplaces, and communities in general can avoid lockdowns. In this work, we analyzed two different sampling methods: oropharyngeal swabs and saliva collection. Oropharyngeal swabs must be collected by trained health personnel at clinics, whereas self-assisted saliva collection can be performed at any given location. It was found that the two sampling methods were comparable. Saliva sampling is a simple method that allows easy mass testing using minimal resources from the existing health care system, and this method may therefore prove to be an effective tool for containing the COVID-19 pandemic.

-Line 19: "It was found that pure saliva kept the virus stable for 9 days...". Stability of a virus is usually measured by their viability to infect cells. The use of the word "stable" in the context of the work presented in the manuscript is therefore incorrect and this issue should be addressed all over the text; -the introduction could be more balanced displaying more information regarding the core topic of the manuscript and less in areas that are not that relevant for the work that is presented. For instance, the authors present nothing less than 6 bibliographic references (7-12) concerning the Omicron variant and very few references concerning works that are already published and that are very similar to the one that is presented, i.e. testing the performance of saliva as a specimen to detect SARS-CoV-2 instead of NPS or OPS. An inversion of this situation would enrich the introduction; -Line 89 -Buffers 1 and 2 are referenced using the catalog number but this is incomplete as the manufacturer name is not mentioned; -Line 99 -the title of this section (2.2) is not adequate. It should be more explicit. The section should also have the PCR conditions that have been used because this is the basis of the work performed; Line 100 -how was the sample homogeneization performed? Line 141 -Title of section 2.5.1 is not correct. The preservation of the virus SARS-CoV-2 is not the subject of the study. Otherwise the authors should report results on the infectivity of samples; Lines 142-143 -the authors state that the "The cohort comprises seven cases which had all been confirmed positive by RT-PCR earlier the same day in February 2021." What type of specimens were used to confirm the samples positivity? Line 144 -The authors state that "The saliva samples were mixed with Buffer 1 and RT-PCR was performed.". No RNA extraction was performed after mixing the samples with Buffer 1? Line 147 -What type of specimens were used to confirm the samples positivity? Lines 150-154 -the authors state that "Over a period of nine days, RT-PCR was performed...". Please clarify: during this period the each sample was subjected to several extractions? (one extraction for every RT-PCR analysis?); Line 171 -Title of this section is not correct. The authors are not analysing the SARS-CoV-2 preservation. Should be corrected. Line 183-185 -The authors state that "The Ct values for the SARS-CoV-2 specific genes were on average increased by 2.5 cycles for pure saliva stored at 4{degree sign}C and by 3.9 cycles when stored at RT during a time span of nine days.". However, in the material and methods section and also in the caption of figure 2, the authors refer to 3 specific genes (IP2, IP4 and E); Line 209 and others -please clarify how was calculated the specificity; Lines 240-242 -the phenomenon is presented as a bias but is perfectly normal and commonly observed in multiplex reactions; The manuscript deals with human samples but there are no reference to an approval of the work by an ethic commission nor the consent of the participants for the study.
Reviewer #2 (Comments for the Author): well controlled study with two samples types, those conclusions for saliva based COVID testing from previous studies are mixed, this study indeed gave some very positive conclusion for saliva samples, this may relate to oropharyngeal swab, different from nasopharyngeal swab.

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Response to Reviewers
Reviewer #1: The manuscript "Comparison of SARS-CoV-2 Analysis from Saliva Sampling and Oropharyngeal Swab" aims to compare the SARS-CoV-2 molecular detection performance using two different specimens which present significant diferences in terms of sample collection difficulty. The manuscript is interesting as all data that may contribute to the development of alternative methods that facilitate viral detection is relevant. Specific comments on the manuscript: -the title is not entirely correct. In fact, the manuscript does not deal with the analysis of SARS-CoV-2 but with its detection in two different matrices. As a sugestion the word "analysis" should be replaced by "detection; We agree with you. The title has been changed. All added words in the manuscript have been marked with yellow. All deleted words in the manuscript have been marked with red and strikethrough.
-Line 19: "It was found that pure saliva kept the virus stable for 9 days..

.". Stability of a virus is usually measured by their viability to infect cells. The use of the word "stable" in the context of the work presented in the manuscript is therefore incorrect and this issue should be addressed all over the text;
We see your point in line 19, as it was written as viral stability and not viral RNA stability. We have changed the wording of line 19 as well as headline 2.5.1 and 3.1. However, we have to disagree regarding the use of the word "stable" in the context of SARS-CoV-2 RNA stability as we find it suitable. Referring to the Medical Subject Headings (MeSH), the definition of RNA Stability is "The extent to which an RNA molecule retains its structural integrity and resists degradation by RNASE, and base-catalyzed HYDROLYSIS, under changing in vivo or in vitro conditions" 1 , and we find it to fit the subject matter of the work.
-the introduction could be more balanced displaying more information regarding the core topic of the manuscript and less in areas that are not that relevant for the work that is presented. For instance, the authors present nothing less than 6 bibliographic references (7-12) concerning the Omicron variant and very few references concerning works that are already published and that are very similar to the one that is presented, i.e. testing the performance of saliva as a specimen to detect SARS-CoV-2 instead of NPS or OPS.

An inversion of this situation would enrich the introduction;
We see your point about the balance of the core topic and the section about the Omicron variants. It was written in the first place to emphasize that SARS-CoV-2 testing is still important although the infectious rate at the time was not that high. Adjustments have been made to the introduction by removing some of the less relevant parts.
-Line 89 -Buffers 1 and 2 are referenced using the catalog number but this is incomplete as the manufacturer name is not mentioned; Thank you for noticing. The manufacturer has been added.
-Line 99 -the title of this section (2.2) is not adequate. It should be more explicit. The section should also have the PCR conditions that have been used because this is the basis of the work performed; has now been corrected. In addition, the confidence intervals have been calculated anew using the Wilson Score method to consider the binomial distribution of the data, which we did not do before.

Lines 240-242 -the phenomenon is presented as a bias but is perfectly normal and commonly observed in multiplex reactions;
We agree. It has been deleted.
The manuscript deals with human samples but there are no reference to an approval of the work by an ethic commission nor the consent of the participants for the study. The first line in the methods section mentions that written consent was obtained from each participant before performing the saliva sampling. Furthermore, our study was conducted prior to 26th May 2022. Thus, by Danish national law, the study did not need ethical approval as the study was non-invasive, and it was not an interventional clinical performance study.

Reviewer #2:
"well controlled study with two samples types, those conclusions for saliva based COVID testing from previous studies are mixed, this study indeed gave some very positive conclusion for saliva samples, this may relate to oropharyngeal swab, different from nasopharyngeal swab. this study will add knowledge about these two samples methods, it has merits on diagnostic testing." Thank you for your feedback -We appreciate it.